| Assay Method Information | |
| | KINOMEscan Kinase Assay |
| Description: | Kinase-tagged T7 phage strains are prepared in an E. coli host derived from the BL21 strain. E. coli are grown to log-phase and infected with T7 phage, and incubated with shaking at 32° C. until lysis. The lysates are centrifuged and filtered to remove cell debris. Alternatively, some kinases are produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads are treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads are blocked with excess biotin and washed with blocking buffer (Sea Block, 1% BSA, 0.05% Tween-20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions are assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% Sea Block, 0.17×PBS, 0.05% Tween-20, 6 mM DTT). Test compounds are prepared as 111× stocks in DMSO. Kd values are determined using an 11-point, 3-fold compound dilution series with three DMSO control points. Compounds are distributed by non-contact acoustic transfer and are then directly diluted into the assay for a final concentration of DMSO of 0.9%, with a final volume in each well of 0.02 mL. Assay plates are incubated at room temperature with shaking for one hour, and the affinity beads are then washed with wash buffer (1% PBS, 0.05% Tween-20). The beads are then resuspended in elution buffer (lx PBS, 0.05% Tween-20, 0.05 μM non-biotinylated affinity ligand) and incubated with shaking for 30 minutes. The concentration of kinase in the eluates is measured by qPCR. Binding constants (Kd) are calculated with a standard dose-response curve using the Hill equation, fitted using non-linear least squares fit with the Levenberg-Marquedt algorithm. |
| Affinity data for this assay | |
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